Microscope assemblage

ABSTRACT

A microscope assemblage, in particular for confocal scanning microscopy, having a light source ( 1 ) for illuminating a specimen ( 6 ) to be examined and at least one fluorescent-light detector ( 11, 14 ) for the detection of fluorescent light ( 10, 13 ) generated in the specimen ( 6 ) and at least one transmitted-light detector ( 16 ) for the detection of transmitted light ( 15 ) passing through the specimen ( 6 ), is configured and developed, with a view toward reliable performing a wide variety of experiments with a high level of detection in each case, such that the fluorescent-light and transmitted-light detectors ( 11, 14; 16 ) are arranged in such a way as to make possible simultaneous detection of fluorescent and transmitting light ( 10, 13; 15 ).

CROSS REFERENCE TO RELATED APPLICATIONS

This invention claims priority of a German patent application DE 100 03570.1 which is incorporated by reference herein.

FIELD OF THE INVENTION

The invention concerns a microscope assemblage, in particular forconfocal scanning microscopy, having a light source for illuminating aspecimen to be examined and at least one fluorescent-light detector forthe detection of fluorescent light generated in the specimen and atleast one transmitted-light detector for the detection of transmittedlight passing through the specimen.

BACKGROUND OF THE INVENTION

Microscope assemblages of the kind cited above are known from practicaluse and exist in a wide variety of embodiments. One example of such amicroscope assemblage is constituted by a confocal scanning microscopein which a specimen to be examined is scanned with a light beam. Themicroscope generally comprises a light source and a focusing opticalsystem with which the light of the source is focused onto an aperturestop. A beam splitter, a scanning device for beam control, a microscopeoptical system, a detection stop, and detectors for the detection ofdetected or fluorescent light are provided.

The illuminating light is usually coupled in via the beam splitter. Thefocus of the light beam is moved with the scanning device in onespecimen plane. This is usually done with two mirrors which are tilted,the deflection axes usually being located perpendicular to one another,so that one mirror deflects in the X direction and the other in the Ydirection. Tilting of the mirrors is brought about, for example, withthe aid of galvanometer positioning elements. In this “descan”arrangement that is usual, the fluorescent or reflected light comingfrom the specimen arrives via the same scanning mirror back at the beamsplitter and passes through it, then being focused onto the detectionstop behind which the detectors are located. Detected light that doesnot derive directly from the focus region takes a different light pathand does not pass through the detection stop, so that a point datum isobtained and yields, by scanning of the specimen, a three-dimensionalimage. Illumination and detection occur here on the objective side, i.e.by way of the microscope optical system.

In a transmitted-light arrangement it is also possible, for example, todetect the fluorescent light or the transmitted light (the transmittedexciting light) on the condenser side, i.e. on the side of a condenserarranged after the specimen. The detected light beam then does not passvia the scanning mirror to the detector. An arrangement of this kind iscalled a “non-descan” arrangement.

In the transmitted-light arrangement, a condenser-side detection stopwould be necessary for detection of the fluorescent light in order, asin the case of the descan arrangement described, to achievethree-dimensional resolution. In the case of two-photon excitation,however, a condenser-side detection stop can be dispensed with, sincethe excitation probability depends on the square of the photon densityor intensity, which of course is much greater at the focus than in theadjacent regions. A very large proportion of the fluorescent light to bedetected therefore derives, with high probability, from the focusregion, rendering superfluous any further differentiation, using a stoparrangement, between fluorescent photons from the focus region andfluorescent photons from the adjacent regions.

Especially given that the yield of fluorescent photons with two-photonexcitation is in any case low, a non-descan arrangement, in which lesslight is generally lost along the detected light path, is of interestBut even when fluorescent light is observed in this manner, celloutlines, for example, cannot be detected sufficiently well because theyare not labeled in living preparations, so that it would be desirablesimultaneously to be able to observe the transmitted light, which wouldallow definite conclusions to be drawn.

Microscope assemblages do exist in which objective-side fluorescencedetection on the one hand, and condenser-side transmission detection onthe other hand, are possible. In this context, however, changing fromobjective-side fluorescence detection to condenser-side transmissiondetection and vice versa requires a mechanical switching operation inwhich mirrors and filters must be mechanically displaced. This can causeshocks to the specimen which destroy it. Experiments with micropipettearrangements, in particular, are thus almost ruled out.

SUMMARY OF THE INVENTION

The present invention is therefore based on the object of describing amicroscope assemblage of the kind cited initially in which a very widevariety of experiments, in particular experiments with micropipettearrangements, can reliably be made with a high level of detection ineach case.

The aforesaid object is achieved by a microscope assemblage whichcomprises: a light source for illuminating a specimen, at least onefluorescent-light detector for the detection of fluorescent lightgenerated in the specimen and at least one transmitted-light detectorfor the detection of transmitted light passing through the specimen,wherein the fluorescent-light and transmitted-light detectors arearranged to enable simultaneous detection of fluorescent and transmittedlight.

The object is achieved as well by a microscope assemblage whichcomprises: a light source for illuminating a specimen, at least onefluorescent-light detector for the detection of fluorescent lightgenerated in the specimen; at least one transmitted-light detector forthe detection of transmitted light passing through the specimen, whereinthe fluorescent-light and transmitted-light detectors are arranged toenable simultaneous detection of fluorescent and transmitted light and afirst polarization device is provided between the light source and thespecimen, a second polarization device in provided after the specimen.

Furthermore, the object is achieved by an other embodiment of themicroscope assemblage, which comprises: a light source for illuminatinga specimen, at least one fluorescent-light detector for the detection offluorescent light generated in the specimen, wherein the specimendefines a top side facing the light source and a bottom side facing awayfrom the light source, at least one transmitted-light detector for thedetection of transmitted light passing through the specimen, and anadditional light source being arranged on the side of the specimenfacing away from the light source.

What has been recognized according to the present invention is that byskillful arrangement of the fluorescent-light and transmitted-lightdetectors, the aforesaid object is achieved in surprisingly simplefashion. For this purpose, the fluorescent-light and transmitted-lightdetectors are arranged in such a way that simultaneous detection offluorescent light and transmitted light is made possible. Switching overbetween transmitted-light and fluorescent-light detection is no longernecessary, so that mechanical shocks to the sample or specimen areprevented. A high level of detection is thus achieved in terms of boththe detection of transmitted light and the detection of fluorescentlight, even with sensitive specimens.

The microscope assemblage according to the present inventionconsequently provides a microscope assemblage with which a very widevariety of experiments, in particular experiments with micropipettearrangements, can reliably be made with a high level of detection ineach case.

BRIEF DESCRIPTION OF THE DRAWINGS

In conjunction with the explanation of the preferred exemplaryembodiments of the invention with reference to the drawings, a generalexplanation is also given of preferred embodiments and developments ofthe teaching. In the drawings:

FIG. 1 shows, in a schematic depiction, a first exemplary embodiment ofa microscope assemblage according to the present invention;

FIG. 2 shows, in a schematic depiction, a second exemplary embodiment ofa microscope assemblage according to the present invention having twopolarization prisms;

FIG. 3 shows, in a schematic depiction, a third exemplary embodiment ofa microscope assemblage according to the present invention having asector stop; and

FIG. 4 shows, in a schematic depiction, a fourth exemplary embodiment ofa microscope assemblage according to the present invention having anadditional light source.

FIG. 5 illustrates an embodiment of the present invention comprising anadditional light source and a transmitted light detector operativelyarranged on the side of the specimen facing away from the light source1.

FIG. 6 illustrates an embodiment of the present invention comprising adetector for detecting both transmitted and fluorescent light in asingle detector.

DETAILED DESCRIPTION OF THE INVENTION

In a concrete embodiment, at least one fluorescent-light detector couldbe arranged on the side of the specimen facing away from the lightsource. Alternatively or in addition thereto, at least onetransmitted-light detector could be arranged on the side of the specimenfacing away from the light source. This makes possible, for example,simultaneous detection of fluorescent light and transmitted light by wayof detectors that are each arranged on the side of the specimen facingaway from the light source. This has the advantage of a well-organizedarrangement of the detectors in one specific region of the microscopeassemblage.

In addition, a condenser for the transmitted light and the fluorescentlight could be arranged on the side of the specimen facing away from thelight source. In the interest of particularly effective lightcollection, the aperture of the condenser could be larger than theaperture of an objective arranged between the light source and thespecimen. When a condenser of this kind is present, and with acorresponding arrangement of the detectors, it is possible to speak of“condenser-side” detection of fluorescent light and transmitted light.

For separate detection of transmitted light and fluorescent light, thetransmitted light and the fluorescent light could be divisible on theside of the specimen facing away from the light source, preferably afterpassing through the condenser. This would result in a physicalseparation of the fluorescent light from the transmitted light Thismakes possible separate measurement of the corresponding light outputsin different detectors.

Concretely, at least one color beam splitter could be used for division.In this context, multiple color beam splitters could be arranged onebehind another in order to allow the separation of different wavelengthsor wavelength regions.

As an alternative to this, at least one semitransparent mirror could beused for division. That mirror or mirrors could have a bandpass filteror blocking filter placed after them. Even when mirrors are used as thedivision component, several such mirrors could be arranged one behindanother, optionally with a bandpass filter or blocking filter placedafter them. This, too, makes it possible to divide the fluorescent lightinto several spectral regions.

As an alternative to the use of color beam splitters or mirrors, amultiband detector that is described, for example, in DE 199 02 625 A1could be used for division. A multiband detector of this kind also makesit possible to divide the fluorescent light into several spectralregions.

In a particularly compact embodiment of the microscope, the fluorescentlight and transmitted light could be detectable in the same detector. Inthe interest of the clearest possible differentiation, however, thefluorescent light and transmitted light could be detectable in differentdetectors.

The microscope assemblage according to the present invention can be usedin particular for interference contrast microscopy. For this purpose, afirst polarization device could be arranged between the light source andthe specimen, preferably before the objective, and a second polarizationdevice after the specimen, preferably after the condenser. Thepolarization devices could be constituted, in particularly simplefashion, by prisms. Wollaston prisms are particularly suitable in thiscontext.

In order to determine whether any rotation of the linear polarizationplane has occurred, a polarization filter could be arranged before thetransmitted-light detector. The polarization filter must be oriented insuch a way that it would block an illuminating light beam without theinfluence of a polarization device.

The microscope assemblage according to the present invention could alsobe used in transmitted-light contrast microscopy. For that purpose, asector optical system, sector polarization optical system, sector stop,sector phase stop, or sector phase filter could be arranged in the beampath. The sector optical system, sector polarization optical system,sector stop, sector phase stop, or sector phase filter could be arrangedin a Fourier plane of the beam path. For that purpose, the sectoroptical system, sector polarization optical system, sector stop, sectorphase stop, or sector phase filter could be arranged in the Fourierplane immediately before the transmitted-light detector. This would makeit possible to implement, for example, the Dodt method or Hoffmanmethod. The fluorescent light could also be simultaneously observed inthis context, although this cannot be done confocally with one-photonexcitation.

With regard to a further particular embodiment of the microscopeassemblage, an additional light source could be arranged on the side ofthe specimen facing away from the light source (on the condenser side,if a condenser is present). As a result, the specimen could optionallybe illuminated on the condenser side, and detection could then takeplace on the side of the specimen facing toward the light source (theobjective side). The additional light source could, in simple fashion,be a white light source.

For phase contrast, a sector optical system, sector polarization opticalsystem, sector stop, sector phase stop, or sector phase filter could beassociated with the additional light source. In particularly favorablefashion, the sector optical system, sector polarization optical system,sector stop, sector phase stop, or sector phase filter could be arrangedin the Fourier plane before the additional light source.

A scanning device could be arranged on the side of the specimen facingtoward the light source. The light generated by the additional lightsource could also be deflected by the scanning device before it strikesa transmitted-light detector or scanner detector. For this purpose, atleast one transmitted-light detector could be arranged on the side ofthe specimen facing toward the light source, preferably on the side ofthe scanning device facing away from the specimen.

In particularly advantageous fashion, a laser could be used as the lightsource. The use of other suitable light sources is also conceivable,however.

The microscope assemblage according to the present invention is suitablein particular for the simultaneous detection of one or more wavelengthregions of the fluorescent light generated by multiple-photon excitationand/or of the fluorescent light generated by one-photon excitation orsecond harmonic generation (SHG), and of transmitted light.

In a specific application, the microscope assemblage according to thepresent invention could also be used to detect fluorescent light ofdifferent wavelength regions and transmitted light sequentially and notsimultaneously, with no need for mechanical switchover operations (e.g.the displacement or replacement of beam splitters or filters) whichinevitably cause shocks to the specimen. The microscope assemblage istherefore also particularly suitable for sequential detection ofdifferent fluorescent spectral regions and/or of transmitted light inapplications with specimens that are externally influenced, for exampleby micromanipulators, micropipettes, or the like.

FIG. 1 shows, in a schematic depiction, a first exemplary embodiment ofa microscope assemblage according to the present invention. Themicroscope assemblage is a confocal laser scanning microscope. Themicroscope has a light source 1 configured as a laser. Light source 1emits an illuminating light beam 2 that is reflected via a main beamsplitter 3 to a scanning device 4. Scanning device 4 guides illuminatinglight beam 2 by way of a microscope optical system or objective 5through a specimen 6. Both the transmitted light passing throughspecimen 6 and the fluorescent light generated in specimen 6 pass via acondenser 7 and a deflection mirror 8 to a first color beam splitter 9which splits off the spectrally lower-wavelength region 10 of thefluorescent light and reflects it to a fluorescent-light detector 11.The spectrally higher-wavelength region 13 of the fluorescent light isreflected via a color beam splitter 12 to a further fluorescent-lightdetector 14. Transmitted light 15 arrives at a transmitted-lightdetector 16 arranged in the straight-ahead direction.

The microscope assemblage consequently has a light source 1 forilluminating a specimen 6 being examined, two fluorescent-lightdetectors 11 and 14 for detecting fluorescent light 10 and 13 generatedin specimen 6, and a transmitted-light detector 16 for detectingtransmitted light 15 passing through specimen 6. The fluorescent-lightand transmitted-light detectors 11, 14, and 16 are arranged in such away as to make possible simultaneous detection of fluorescent andtransmitted light 10, 13, and 15. The specimen 6 defines a top side 6 afacing the light source 1 and a bottom side 6 b facing away from thelight source 1.

The microscope assemblage shown in FIG. 1 furthermore has a detector 17that is arranged on the objective side. The detector 17 in thisembodiment ist used for the detection of fluorescent light from thespecimen 6.

Both fluorescent-light detectors 11 and 14 are arranged on the side ofspecimen 6 facing away from light source 1. Transmitted-light detector16 is also arranged on the side of specimen 6 facing away from lightsource 1.

FIG. 2 shows, in a schematic depiction, a second exemplary embodiment ofa microscope assemblage according to the present invention. Themicroscope assemblage shown in FIG. 2 corresponds substantially to themicroscope assemblage of FIG. 1, components that correspond tocomponents already described in FIG. 1 being labeled with the samereference characters.

The microscope assemblage shown in FIG. 2 is suitable in particular fordifferential interference contrast (DIC) microscopy with simultaneousfluorescence detection. For that purpose, the microscope assemblage hastwo polarization devices configured as polarization prisms 18.Polarization prisms 18 are constituted by so-called Wollaston prisms.The one polarization prism is arranged between light source 1 andspecimen 6, more precisely before objective 5. The second polarizationprism 18 is arranged after specimen 6, more precisely after condenser 7.

Differential interference contrast makes it possible to observe phaseobjects with simultaneous fluorescence detection. The linearly polarizedexciting light of light source 1 (configured as a laser) is split intotwo partial beams, before objective 5, with the aid of the firstpolarization prism 18. The two partial beams then pass through specimen6 along slightly different paths, and are recombined after condenser 7with the aid of the second polarization prism 18. If the two partialbeams have traveled along optical paths of different lengths, this isexpressed, after the beams are combined, in a rotation of the linearpolarization plane which is analyzed by way of a polarization filter 19before transmitted-light detector 16. Polarization filter 19 must beoriented in such a way that it would block an illuminating beam passingdirectly through without polarization prisms 18.

FIG. 3 shows, in a schematic depiction, a third exemplary embodiment ofa microscope assemblage according to the present invention. Themicroscope assemblage shown here corresponds substantially to themicroscope assemblage shown in FIG. 1, components that correspond tocomponents already described in FIG. 1 being labeled with the samereference characters.

The microscope assemblage shown in FIG. 3 can be used in particular forphase contrast microscopy. For that purpose, the microscope assemblagehas a sector stop 20 arranged in the Fourier plane beforetransmitted-light detector 16. Fluorescent light 10 and 13 issimultaneously observable.

The Dodt method can be performed with sector stop 20. Also usable, as analternative to a sector stop 20, is a sector polarization optical systemwith which, for example, the Hoffman method can be used, fluorescentlight 10 and 13 being simultaneously observable. With one-photonexcitation, however, fluorescent light 10 and 13 cannot be observedconfocally.

FIG. 4 shows, in a schematic depiction, a fourth exemplary embodiment ofa microscope assemblage according to the present invention. Themicroscope assemblage shown in FIG. 4 corresponds for the most part tothe microscope assemblage shown in FIG. 1, components that correspond tocomponents already described in FIG. 1 being labeled with the samereference characters.

In the microscope assemblage shown in FIG. 4, specimen 6 is alsoilluminated on the condenser side by way of an additional light source21. Additional light source 21 is thus arranged on the side of specimen6 facing away from light source 1. An additional reverse beam path isthus present here.

Additional light source 21 has associated with it, for phase-contrastpurposes, a sector stop 20 in the Fourier plane in front of additionallight source 21. Additional light source 21 generates an illuminatinglight beam 22 that passes through specimen 6 and is detected by way ofdetector 17. Prior to detection, illuminating light beam 22 experiencesa scanning operation by way of scanning device 4. The detector 17detects the transmitted light and could also be referred to as a“scanner detector.” This detector 17 is capable, for example, of sensingthe Dodt light or Hoffman light.

As shown in FIG. 5, the microscope assemblage of the present inventionmay also be configured to comprise transmitted-light detector 16 andadditional light source 21 operatively arranged on the side of thespecimen 6 facing away from light source 1.

Finally, FIG. 6 illustrates a microscope assemblage according to thepresent invention comprising detector 23 for detecting both transmittedand fluorescent light in a single detector.

The microscope assemblage according to the present invention could beused in particular with two-photon excitation, since in this case anydifferentiation, using a stop arrangement, between fluorescent photonsfrom the focus region and fluorescent photons from the adjacent regionsis superfluous. The microscope assemblage is moreover suitable forinterference contrast microscopy using polarization prisms, and fortransmitted-light contrast microscopy with the aid of sector stops.

The microscope assemblage can be used for sequential detection ofdifferent fluorescent spectral regions and/or of transmitted light,without mechanical switchover operations. The microscope assemblage isthus particularly suitable for applications with micropipettes,micromanipulators, or the like.

Regarding further advantageous embodiments of the microscope assemblageaccording to the present invention, reference is made, in order to avoidrepetition, to the general portion of the specification and to theappended Claims.

Lastly, be it noted expressly that the above-described exemplaryembodiments of the microscope assemblage according to the presentinvention serve only to present the teaching that is claimed, but do notlimit it to those exemplary embodiments.

PARTS LIST

1 Light source

2 Illuminating light beam

3 Main beam splitter

4 Scanning device

5 Objective

6 Specimen

6 a top side

6 b bottom side

7 Condenser

8 Mirror

9 Color beam splitter

10 Wavelength region

11 Fluorescent-light detector

12 Color beam splitter

13 Wavelength region

14 Fluorescent-light detector

15 Transmitted light

16 Transmitted-light detector

17 Detector

18 Polarization prism

19 Polarization filter

20 Sector stop

21 Additional light source

22 Illuminating light beam

23 Transmitted and Fluorescent light detector

What is claimed is:
 1. A microscope assemblage for confocal scanningmicroscopy comprising: a light source (1) for illuminating a specimen(6); at least one fluorescent-light detector (11, 14) for the detectionof fluorescent light (10, 13) generated in the specimen (6), wherein thespecimen (6) defines a top side (6a) facing the light source (1) and abottom side (6 b) facing away from the light source (1); at least onetransmitted-light detector (16) for the detection of transmitted light(15) passing through the specimen (6); said transmitted light comprisingthat light not produced by the fluorescence of said specimen; and, anadditional light source (21) operatively arranged on the side of thespecimen (6) facing away from the light source (1) and arranged forilluminating said specimen; said light source (1) operatively arrangedon a top side of said specimen, said additional light source (21) andsaid transmitted light detector (16) on the side facing away from saidspecimen operatively arranged to simultaneously detect said transmittedlight and to illuminate said specimen.
 2. The microscope assemblage asdefined in claim 1, characterized in that the additional light source(21) is a white light source.
 3. The microscope assemblage as defined inclaim 1, characterized in that an optical system is a member selectedfrom the group consisting of a sector optical system, a sectorpolarization optical system, a sector stop, a sector phase stop and asector phase filter, said optical system associated with said additionallight source.
 4. The microscope assemblage as defined in claim 3,characterized in that the optical system is arranged in a Fourier planebefore the additional light source (21).
 5. The microscope assemblage asdefined in claim 1, characterized in that a condenser (7) for thetransmitted light (15) and the fluorescent light (10, 13) is arranged onthe side of the specimen (6) facing away from the light source (1). 6.The microscope assemblage as defined in claim 5, characterized in thatan objective (5) is arranged between the light source (1) and thespecimen (6) and the aperture of the condenser (7) is larger than theaperture of the objective (5).
 7. The microscope assemblage as definedin claim 6, characterized in that the transmitted light (15) and thefluorescent light (10, 13) are divisible on the side of the specimen (6)facing away from the light source (1), after passing through thecondenser (7).
 8. The microscope assemblage as defined in claim 7,characterized in that at least one color beam splitter (9, 12) is usedto provide light to at least one fluorescent-light detector (11, 14). 9.The microscope assemblage as defined in claim 7, characterized in that amultiband detector is used for spectral separation.
 10. The microscopeassemblage as defined in claim 1, characterized in that the fluorescentlight (10, 13) and transmitted light (15) are detectable in onedetector.
 11. The microscope assemblage as defined in claim 1,characterized in that the fluorescent light (10, 13) and transmittedlight (15) are detectable in different detectors (11, 14; 16).
 12. Themicroscope assemblage as defined in claim 1, characterized in that ascanning device (4) is arranged on the side of the specimen (6) facingtoward the light source (1).
 13. The microscope assemblage as defined inclaim 1, characterized in that at least one detector (17) is arranged onthe side of the specimen (6) facing toward the light source (1), on theside of scanning device (4) facing away from the specimen (6).
 14. Themicroscope assemblage as defined in claim 1, characterized in that thelight source (1) is a laser.